In recent years, a number of investigations carried out on the passive ion permeability of erythrocyte membranes have revealed that a fraction of the ouabain-resistant K+ fluxes is insensitive to Na+ and depends on the presence of Cl-. The transport mechanism underlying the ouabain-, and Na+-insensitive, Cl--dependent K+ flux has been defined as the K-Cl cotransporter. Due to the direction of KCI gradients, K-Cl cotransport when activated induces a loss of KCI and a cell volume reduction. Since a reduction in cell volume is a well established event in red blood cell maturation and K-Cl fluxes through the K-Cl cotransporter are high in reticulocytes and almost absent in mature erythrocytes, it has been hypothesized that the pathway plays a significant role in the maturation process. The kinetic details and the regulation of the pathway have been extensively studied these last few years, but its molecular identity is still unknown. This proposal targets two major objectives: 1) To test the hypothesis that K-Cl cotransport participates in the cell volume reduction occurring through the overall maturation process. We propose to characterize the role of Cl--dependent K+ transport together with other K+ transport systems from undifferentiated cells to reticulocytes by using a mouse erythroleukemia (MEL) cell line (nucleated as opposed to mature mammalian red cells). 2) To clone and identity the transport protein which mediates the coupled K-Cl fluxes. Since trout hepatocytes present the highest level of K-Cl cotransport activity, and since total RNA isolated from trout liver and injected to Xenopus oocytes lead to expression of a Cl-dependent K+ flux, we propose to identity the trout liver cDNA encoding the K-Cl cotransporter. Once this cDNA clone is isolated, we propose to clone the mammalian homologue and initiate a comparative study of mRNA levels, K-Cl cotransport activity, and regulation in the different stages of MEL cell differentiation. Finally, a structure/function study is proposed to uncover the sites of ion binding and to determine whether the N-ethylmaleimide activation occurs through binding to the transport molecule or not. Results of these studies would explain part of the physiological mechanisms leading to the recruitment and/or disappearance of K-Cl cotransport units, or leading to the activation/inactivation process occurring during red cell maturation. Finally, these studies will shed some light on the abnormal activation of this transport pathway in mature human red cells containing hemoglobin S or C.